Post-transcriptional regulation of messenger RNA (mRNA) stability and translation is an important mechanism for rapidly controlling gene expression in response to stimuli, including environmental changes. This project seeks to generate and utilize structural information to enhance our understanding of these processes with an emphasis on the importance of RNA target specificity for proper gene regulation. PUF proteins are sequence-specific RNA-binding proteins that are important regulators of gene expression for embryonic development and germline stem cell maintenance. Beginning with determining the first crystal structure of a PUF protein in complex with RNA to publishing work this year on the specificity of human, yeast, and C. elegans proteins (refs. 1, 2), we have identified both common and unique features of RNA recognition by this family of proteins. The combination of the features in any particular protein results in a unique network of mRNAs that are regulated by that protein. In this fiscal year, we have advanced this work by identifying and studying a conserved feature among a set of PUF proteins. This feature, called an upstream cytosine binding pocket, adds RNA target specificity to these proteins. This additional binding pocket is necessary for highest affinity RNA binding and RNA target selection. However, the position and quantitative contribution of the upstream C varies betweens proteins. Furthermore, we have identified regions in PUF proteins that are critical for RNA binding by mutating systematically the RNA-interacting residues in each repeat for C. elegans FBF-2 and yeast Puf3p and Puf4p (ref. 2). These studies showed that the N- and C-terminal regions are critical for binding affinity with intervening regions allowing diversity of specificity. This conclusion is consistent with the information from our previous structural and functional analyses. We refer to this as a two-handed model of interaction. With this model in mind, we studied the effect of variability of the intervening regions of PUF proteins and RNA target sequences on regulatory specificity. We found that yeast Puf4p binds to two different sets of target sequences, which broadens its known specificity. One set of sites is regulated only by Puf4p, but the second set may be dually regulated by Puf4p or a second yeast PUF protein, Mpt5p.